One-component tissue adhesive and a process for the production thereof

ABSTRACT

A description is given of a one-component tissue adhesive containing, in aqueous solution, fibrinogen, F XIII, a thrombin inhibitor, prothrombin factors, calcium ions and, where appropriate, a plasmin inhibitor and of a process for the production thereof. 
     This adhesive can be reconstituted from a freeze-dried form with water. It can contain all active substances in pasteurized form and is then free of the risk of transmission of hepatitis and HTLV III.

This application is a continuation of application Ser. No. 07/835,118,filed Feb. 14, 1992, now abandoned, which is a continuation of Ser. No.07/069,199, filed Jul. 2, 1987, now abandoned.

The invention relates to a tissue adhesive which contains fibrinogen,factor XIII, a thrombin inhibitor, prothrombin factors, calcium ionsand, where appropriate, a plasmin inhibitor. The accelerators which arenaturally present on the wound which is to be bonded result in thethrombin which is necessary for adhesion being liberated from theprothrombin in the adhesive.

The physiological processes involved in the repair of lesions in humanor animal tissues result in fibrin being deposited in the region of theinjury. This is initiated by thromboplastin flowing out of the injuredcells and, on contact with plasma factor VII forming factor X activatorwhich then, together with factor V and in a complex with phospholipidsand calcium, converts prothrombin into thrombin. The action of thrombinin turn results, via elimination of the fibrinopeptides A and B fromfibrinogen, in fibrin monomers which aggregate to form fibrin filamentswhich are covalently cross-linked, with the formation of isopeptidebonds by F XIIIa, a transglutaminase which is likewise activated bythrombin. An inhibitor, namely alpha₂ -antiplasmin, is bound by factorXIII onto the fibrin filaments and protects them from degradation byplasmin.

It is evident from this process of hemostasis and wound healing that, inaddition to fibrinogen as substrate, two enzymes are required: thrombinand factor XIII.

The task of thrombin in this connection is to activate both fibrinogenand factor XIII, that is to say to convert them into their reactiveform. This is the fibrin monomer in the case of fibrinogen, and is thecatalytically active transglutaminase (F XIIIa) in the case of factorXIII.

European Patent 0,068,047 discloses an enriched plasma fraction which issuitable as a wound closure and contains fibrinogen, a fibrinolysisinhibitor and thrombin or prothrombin in an anhydrous system. Thisadhesive cannot be applied dissolved in water, but can be applied onlyas a powder, which is a disadvantage for use. When the mixture isdissolved in water, the components react together, and a clot isproduced.

For a one-component adhesive with water as solvent it is difficult tocombine the active substances in such a way and in such quantitativeratios that no premature and undesired activation takes place during theproduction of the adhesive, the reconstitution with a solvent and themaintenance in readiness for use and, on the other hand, a sufficientlyhigh strength is achieved relatively quickly after application to theadhesion site.

Surprisingly, conditions allowing reproducible production of aone-component adhesive, which can be manipulated in practice, in aphysiological medium have been found.

The requirements for a one-component adhesive of this type to beproduced and be amenable to processing are that the relativeconcentrations of fibrinogen, plasmin inhibitor, F XIII, prothrombinconcentrate, thrombin inhibitor and calcium ions are optimized. Thisparticularly applies to AT III as thrombin inhibitor and to calciumions, since the calcium ions are essential for activation of thecoagulation factors. High concentrations increase the rate of thrombinformation, and low concentrations slow it down. Hence it is necessarythat the concentration of calcium ions is optimal. Since, when this isthe case, the formation of thrombin cannot be ruled out, it is expedientto combine with a thrombin inhibitor, preferably AT III. This isbecause, on the one hand, the formation of thrombin must be ruled outduring the production of the adhesive and, on the other hand, theadhesive must rapidly form a stable clot when coagulation, that is tosay the deposition of a fibrin film, is initiated on contact with woundsurfaces. Accordingly, the production of a thrombin-containingone-component adhesive and its use in aqueous solution is inconceivableand, moreover, it is possible to use prothrombin only under conditionswhich prevent activation to thrombin during production and preparationfor use. These include setting up defined concentrations of thrombininhibitor and calcium ions.

Thus the invention relates to a tissue adhesive containing in aqueoussolution fibrinogen, F XIII, a thrombin inhibitor, prothrombin factors,calcium ions and, where appropriate, a plasmin inhibitor.

It was surprising that it is possible to pack, and use as tissueadhesive, fibrinogen together with prothrombin as a so-calledhypercoagulable solution which, moreover, also contains calcium ions andAT III.

An adhesive of this type according to the invention can be convertedinto a solid form, for example freeze-dried, and reconstituted withwater. It can contain all the active substances in pasteurized form, andis then free of the risk of transmission of hepatitis and HTLV III.

The mixing ratio of the active substances fibrinogen, plasmin inhibitor,F XIII, prothrombin factors, calcium ions and AT III is such that nopremature activation takes place and such that, however, the mixturespontaneously coagulates, and deposits fibrin, on contact with woundsurfaces.

The time in which this takes place is consistent with clinicalrequirements. The one-component adhesive according to the invention doesnot differ in its action from the two-component systems of the state ofthe art.

The mixture of active substances in this adhesive is such that, in thesolution for use, the concentration of fibrinogen is 70-90 mg/ml andthat of the prothrombin factors is between 10 and 30 U/ml based on F II.The concentration of calcium ions, some of which are protein-bound inthe adhesive, is intended to be 0.5-1 mmol/l in the solution for use.Examples of thrombin inhibitors are AT III, hirudin and heparin, and ATIII is preferably used in a concentration of 0.1-1U/ml of tissueadhesive; at this concentration it prevents, at the abovementionedconcentrations of prothrombin factors and calcium ions, premature fibrinformation. On the other hand, however, it is possible to favor thelatter when this appears desirable on the basis of the indication. Forthis purpose, it is possible to dissolve the freeze-dried adhesive in ahigher final concentration of calcium ions than 0.5-1 mmol/l. However,rapid use after reconstitution is then necessary.

To produce the adhesive, the active substances are initially produced insoluble form having the highest possible activity and, whereappropriate, having been pasteurized. The fibrinogen concentrationshould be as high as possible, since the strength of adhesion increaseswith increasing concentration. The fibrinogen can, for example, beproduced and pasteurized as described in European Patent 0,103,196, andgiven the desired solubility by additives of the type of compoundcontaining urea or guanidine residues as described in European Patent0,085,923. Factor XIII can be pasteurized as described in EuropeanPatent 0,018,561, and the prothrombin factors can be pasteurized asdescribed in European Patent 0,056,629.

The active substances in the preferred adhesive according to theinvention in principle comprise human fibrinogen in the highest possibleconcentration, enriched with factor XIII and the factors of theprothrombin complex (factor II, VII, IX and X), calcium ions andantithrombin III, The tissue adhesive also contains an inhibitor whichinhibits plasminogen activators and/or plasmin and thus protects theresulting fibrin from degradation by plasmin. This inhibitor may be inthe form of a concomitant substance of the fibrinogen selected for thebonding, but is preferably added to the adhesive in the form ofaprotinin. To improve the reconstitution of the tissue adhesive afterfreeze-drying, it is possible for the latter to contain albumin and/orsubstances which contain the urea or guanidine residue and also contain,where appropriate, an amino acid with a hydrophobic side-chain or awater-soluble fatty acid and, in addition, heparin.

The fibrinogen which is suitable and preferred for preparation of thetissue adhesive is purified and already contains factor XIII. However,apart from this, also suitable is cryoprecipitate which, experience hasshown, contains alpha₂ -antiplasmin and human albumin in addition tofactor XIII. It is also possible, however, to produce an adhesive ofcomparable action by mixing the individual components, namely: humanfibrinogen, factor XIII (from plasma or placenta), plasmin inhibitors,prothrombin factors (factor II, VII, IX and X), albumin, AT III andcalcium ions, a compound with a urea or guanidine residue, or an aminoacid with a hydrophobic side-chain or a water-soluble fatty acid.

The mixture of the active compounds for the adhesive is advantageouslyin the form of a solid, preferably a lyophilizate.

The tissue adhesive preferably contains the following quantities ofactive compounds, which are pasteurized where appropriate, based on 1 mlof solution for use:

65-115, preferably 70-90, mg of highly purified human fibrinogen

40-80 U of factor XIII

1-50, preferably 10-30, IU of PPSB (prothrombin factors) based on F II(prothrombin)

0-10,000 KIU of aprotinin

0.01-50, preferably 0.1-1, IU of antithrombin III

0-5 USP U heparin and

0.1-5, preferably 0.5-1 mmol/l of calcium ions, preferably as calciumchloride.

The adhesive can contain, for example glutamate, L-isoleucine,L-arginine or human albumin as stabilizers and solubilizers.

The mixture of the solid active compounds of the tissue adhesive can bedissolved in water, or in a solvent containing calcium ions, in a shorttime at 20° C. When applied to the tissue which is to be united orbonded the adhesive develops within a sufficiently short time a highbonding strength without thrombin being necessary as a second component.

Accordingly, it is possible to apply the adhesive in liquid form using asingle syringe, so that preparation, manipulation and use arestraightforward. The adhesive is tolerated and is completely absorbed.

This one-component adhesive does not differ, either in the bonding timeor in the rupture strength, from a two-component adhesive, as has beenfound in bonding tests in the model of puncture wounds of the rat skinand in the bonding of small intestinal anastomoses in hogs. It is evenpossible to increase the bonding strength by choosing an optimal calciumion concentration of the solution which is used to dissolve thefreeze-dried active compounds.

The examples which follow are intended to illustrate the invention.

EXAMPLE 1

To produce 500 ml of tissue adhesive, 580 g of pasteurized highlypurified human fibrinogen which contained 2 calcium ions per mole andhad been obtained as described in European Patent 0,103,196 weredissolved in 580 ml of buffer, pH 7.5 (dialysis buffer), with thefollowing composition:

0.05 mol/l NaCl

0.005 mol/l trisodium citrate

0.3 g/100 ml L-arginine monohydrochloride.

The fibrinogen solution which had been obtained in this way was dialysedtwice for 16 hours and once for 8 hours, in each case against 36 l ofbuffer of the same composition, at +4° C. The volume of the fibrinogensolution after the dialysis was 1.53 l. The solution was thencentrifuged at 8000×g for 20 min. The optical density of the solution,measured at 280 nm, was 47. This solution was diluted with theabovementioned buffer, to which the other adhesive components had beenadded (see below), to an optical density (280 nm) of 35 to 37, i.e. toabout 20 g/l human fibrinogen.

The specific procedure for this was as follows: the dialysed andcentrifuged human fibrinogen solution (1.53 l) was filtered successivelythrough filters of pore sizes 0.65 and 0.2μ at 35° to 37° C. 117.5 ml ofpasteurized F XIII concentrate, containing 350 U of F XIII/ml ofphysiological saline solution, was filtered through a filter of 0.2μpore size into the resulting sterile fibrinogen solution.

Then 515,000 KIU (kallikrein inhibitor units) of aprotinin, 5.13 g of Naglutamate, 33.9 ml of a human albumin solution containing 20 g/100 ml,6.78 g of isoleucine and 10,000 U of pasteurized PPSB concentrate(prothrombin factor concentrate; activity in units based on F II), 100 Uof AT III (pasteurized) and 3.1 ml of 0.1 mol/l CaCl₂ solution wereadded.

The volume was then made up to 407.5 ml with dialysis buffer. Thissolution was filtered through a filter of 0.2μ pore size into the FXIII-containing fibrinogen solution. About 2,055 ml of solution of pH7.5 and the following composition were obtained:

    ______________________________________                                        human fibrinogen                                                                            2.0      g/100 ml                                               human albumin 0.33     g/100 ml                                               arginine      0.3      g/100 ml                                               Na glutamate  2.5      g/l                                                    isoleucine    3.3      g/l                                                    factor XIII   20,000   U/l                                                    aprotinin     250,000  KIU/l                                                  prothrombin   5,000    U/l (calculated as F II)                               antithrombin III                                                                            50       U/l                                                    NaCl          0.05     mol/l                                                  trisodium citrate                                                                           0.005    mol/l                                                  calcium chloride                                                                            0.15     mmol/l.                                                ______________________________________                                    

The sterile solution was packed in 4 ml portions and freeze-dried. Acolorless, rapidly dissolving lyophilizate was obtained.

For use as tissue adhesive, one packed portion was taken up in 1 ml ofsolvent.

EXAMPLE 2

To produce 125 ml of tissue adhesive, 100 g of a pasteurized, highlypurified human fibrinogen fraction, obtained as described in EuropeanPatent 0,103,196, were dissolved in 112 ml of dialysis buffer anddialyzed three times against 10 l each time of the same buffer as inExample 1. The volume of the concentrated fibrinogen solution afterdialysis was 269 ml. It was centrifuged at 8,000×g for 20 min. Theoptical density of the solution at 280 nm was 69.2. The solution wasdiluted with a buffer whose composition corresponds to that of thedialysis buffer, and in which all the other adhesive components havebeen mixed, in such a way that an optical density at 280 nm of 35-37,based on fibrinogen, was reached.

The procedure for this was as follows:

127 ml of buffer were introduced into a vessel and then 8.2 ml of humanalbumin solution containing 20 g of human albumin per 100 ml ofsolution, 6 ml aprotinin amounting to 125,000 KIU and 50 ml ofprothrombin concentrate containing 2,500 U of factor II and 25 U of ATIII were added. 1.65 g of L-isoleucine and 1.25 g of Na glutamate weredissolved in this, and then 40 ml of F XIII concentrate containing10,000 U of F XIII were added, the mixture was homogenized, and the pHof the solution was adjusted to pH 7.5.

Subsequently this solution was mixed and homogenized with the fibrinogensolution which is described above and 0.75 ml of a 0.1 molar CaCl₂solution, and was sterilized by filtration through a filter of pore size0.2μ at 35°-37° C. About 500 ml of sterile solution of the compositionas in Example 1 were obtained. This solution was packed in 4 mlquantities and freeze-dried. A colorless, rapidly dissolvinglyophilizate was obtained.

For use as tissue adhesive, one packed portion is taken up in 1 ml ofsolvent.

We claim:
 1. A one component tissue adhesive in solid form having asactive substances when reconstituted in 1 ml of solution:65-115 mg ofhuman fibrinogen, 40-80 U of factor XIII, 1-50 IU of PPSB (prothrombinfactors) based on factor II (prothrombin), 0.01-50 IU of antithrombinIII, 0.1-5 mmol/l of calcium ions, and 1-10,000 KIU of aprotinin,whereinthe ratio of said active substances is such that there is no prematurecoagulation of said adhesive during preparation, and such thatcoagulation is initiated on contact with a wound surface.